The actions and interactions of ligands that affect the rat hepatic glucagon-sensitive adenylate cyclase system have been investigated. The divalent cations, Mg 2 ion and Mn 2 ion, activate the enzyme system by a mechanism independent of the concentration of uncomplexed ATP in the medium. This rules out models in which the effects of divalent cations are attributed to a lowering of the free, uncomplexed ATP concentration. Two activators of the enzyme system, glucagon and guanine nucleotides, appear to increase the affinity of the system for the cations. Adenosine is a specific inhibitor of the system, and the activating ligands, particularly divalent cations and glucagon, increase the affinity for the nucleoside. Knowledge of the interrelationships of these ligands permits interpretation of kinetic data on adenylate cyclase.